Dimers of Covalent NFKB Inhibitors

ABSTRACT

Provided herein are compounds and methods for modulating the NFκB pathway. More particularly, provided are inhibitors of the NFκB pathway and the uses of such inhibitors in regulating diseases and disorders, e.g., to treat cancer, autoimmune diseases, inflammatory diseases, diabetes, cardiovascular diseases, or neurological diseases.

STATEMENT OF U.S. GOVERNMENT SUPPORT

This invention was made with government support under grant numbers R21CA182820 and R01CA197999 awarded by the National Institutes of Health.The government has certain rights in the invention.

BACKGROUND

Nuclear localization of the transcription factor NFκB has beenimplicated in oncogenesis and drug resistance in a number of cancers.Inhibition of the nuclear translocation of NFκB is hypothesized to be aviable therapeutic approach for cancer, and as such, the development ofdrugs providing novel approaches to inhibiting nuclear localization ofNFκB is becoming an important strategy in addressing the need foradditional therapies for the treatment of these disorders.

SUMMARY

The disclosure provides compounds of Formula I:

wherein X is O, S, or NR⁴;

L is C₁₋₁₈alkylene or C₂₋₁₈alkenylene optionally interrupted with one ormore of (i) non-adjacent heteroatom(s) selected from O, S, and NR⁴, (ii)C(O)NR⁴, (iii) C₆₋₁₀ aryl, (iv) 5-10 membered heteroaryl having 1-4heteroatoms selected from N, O, and S, (v) 3-12 membered cycloalkylring, and (vi) 3-7 membered heterocycloalkyl ring having 1-3 ringheteroatoms selected from O, S, and N, and said aryl, heteroaryl,cycloalkyl, and heterocycloalkyl are optionally substituted with one ormore R⁴;

each R¹ is independently selected from the group consisting of C₁₋₆alkyl, halo, CN, N(R⁴)₂, OR⁴, NO₂, CO₂R⁴, (C═O)R⁴, CON(R⁴)₂, NR⁴(C═O)R⁵,C₆₋₁₀ aryl, and 5-10 membered heteroaryl having 1-4 heteroatoms selectedfrom N, O, and S, and said alkyl can be optionally substituted with 1 to3 R³;

each R² is independently selected from the group consisting of C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, halo, OR⁴, COR⁴, CO₂R⁴, CON(R⁴)₂,N(R⁴)₂, and SR⁴, or a pair of two R² together with the carbon atom towhich they are attached form a saturated or unsaturated 4-8 memberedcycloalkyl or heterocycloalkyl ring, wherein the heterocycloalkyl ringhas 1 or 2 ring heteroatoms selected from O, S, and N, and wherein saidalkyl, alkenyl, alkynyl, cycloalkyl ring, and heterocycloalkyl ring areoptionally substituted with 1 to 3 R³,

with the proviso that at least one R² or one pair of two R² togetherforming a ring comprise an α,β-unsaturated moiety;

each R³ is independently selected from the group consisting of C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, halo, CN, N(R⁴)₂, OR⁴, NO₂, oxo, ═S,═NR⁴, CO₂R⁴, (C═O)R⁴, CON(R⁴)₂, C₆₋₁₀ aryl, and 5-10 membered heteroarylhaving 1-4 ring heteroatoms selected from N, O, and S, and wherein saidalkyl, alkenyl, and alkynyl are optionally substituted with 1 to 3 R⁴;

each R⁴ is independently selected from the group consisting of H, C₁₋₆alkyl, C₂₋₆ alkenyl, and C₂₋₆ alkynyl, wherein said alkyl, alkenyl, andalkynyl are optionally substituted with one or more substituentsselected from the group consisting of halo, CN, NH₂, OH, and C₁₋₆alkoxy; and n is 0-4.

In some embodiments, at least one pair of two R² together with thecarbon atom to which they are attached form a ring having the structure:

wherein * is the carbon atom to which each R² is attached, Z is O orNR⁴, and R⁵ is C₁₋₆alkyl, C₂₋₆alkenyl, C₆₋₁₀ aryl, or 3-7 memberedheterocycloalkyl ring having 1-3 ring heteroatoms selected from O, S,and N.

In various embodiments, L is selected from the group consisting ofuninterrupted C₁₋₁₈alkylene,

Y¹, Y², and Y³ are each independently O or NR⁴, and

indicates that the double bond is cis or trans.

Further provided herein are methods of using the compounds to inhibitthe NFκB pathway. Also provided are methods of treating or preventing adisease or disorder capable of being modulated by NFκB pathwayinhibition.

Other aspects of the disclosure include a compound as disclosed hereinfor use in the preparation of a medicament for treating or preventing adisease or disorder capable of being modulated by NFκB pathwayinhibition in a subject, and the use of a compound as disclosed hereinin a method of treating or preventing a disease or disorder capable ofbeing modulated by NFκB pathway inhibition in a subject

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of Compounds 19, 36-252, and 36-242 on cellgrowth in normal cells (FT28) and ovarian cancer cells (OVCAR5).

FIG. 2 shows the effect of Control (DMSO), flavopirodol, and compound36-252 on apoptosis in five HGSC cell lines as measured by a caspaseassay.

FIG. 3 shows the effect of compounds 36-252 and 36-297 on cancer cells(A549 luciferase cells) as measured by inhibition of TNF-α-induced NFκBactivity.

FIG. 4 shows the results of a Western blot analysis to determinecrosslinking of proteins in the IKK complex.

FIG. 5 shows the results of a side population analysis in OVCAR-5 orA2780 cells.

FIG. 6 shows the results of a toxicity study comparing the effects ofDMSO vs. 100 mg/kg 36-252 on aspartate transaminase (AST) and alaninetransaminase (ALT) in mice.

FIG. 7 shows the results of hematological analysis in control (DMSO) vs.36-252-treated mice.

DETAILED DESCRIPTION

Provided herein are compounds that inhibit the NFκB pathway, and cantreat or prevent a disease or disorder associated with NFκB in asubject. These compounds are useful in the treatment of a variety ofdiseases and disorders, including but not limited to cancer, autoimmunediseases, inflammatory diseases, diabetes, cardiovascular diseases, orneurological diseases.

Compounds of the Disclosure

The disclosure provides compounds of Formula I:

wherein X is O, S, or NR⁴;

L is C₁₋₁₈alkylene or C₂₋₁₈alkenylene optionally interrupted with one ormore of (i) non-adjacent heteroatom(s) selected from O, S, and NR⁴, (ii)C(O)NR⁴, (iii) C₆₋₁₀ aryl, (iv) 5-10 membered heteroaryl having 1-4heteroatoms selected from N, O, and S, (v) 3-12 membered cycloalkylring, and (vi) 3-7 membered heterocycloalkyl ring having 1-3 ringheteroatoms selected from O, S, and N, and said aryl, heteroaryl,cycloalkyl, and heterocycloalkyl are optionally substituted with one ormore R⁴;

each R¹ is independently selected from the group consisting of C₁₋₆alkyl, halo, CN, N(R⁴)₂, OR⁴, NO₂, CO₂R⁴, (C═O)R⁴, CON(R⁴)₂, NR⁴(C═O)R⁵,C₆₋₁₀ aryl, and 5-10 membered heteroaryl having 1-4 heteroatoms selectedfrom N, O, and S, and said alkyl can be optionally substituted with 1 to3 R³;

each R² is independently selected from the group consisting of C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, halo, OR⁴, COR⁴, CO₂R⁴, CON(R⁴)₂,N(R⁴)₂, and SR⁴, or a pair of two R² together with the carbon atom towhich they are attached form a saturated or unsaturated 4-8 memberedcycloalkyl or heterocycloalkyl ring, wherein the heterocycloalkyl ringhas 1 or 2 ring heteroatoms selected from O, S, and N, and wherein saidalkyl, alkenyl, alkynyl, cycloalkyl ring, and heterocycloalkyl ring areoptionally substituted with 1 to 3 R³,

with the proviso that at least one R² or one pair of two R² togetherforming a ring comprise an α,β-unsaturated moiety;

each R³ is independently selected from the group consisting of C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, halo, CN, N(R⁴)₂, OR⁴, NO₂, oxo, ═S,═NR⁴, CO₂R⁴, (C═O)R⁴, CON(R⁴)₂, C₆₋₁₀ aryl, and 5-10 membered heteroarylhaving 1-4 ring heteroatoms selected from N, O, and S, and wherein saidalkyl, alkenyl, and alkynyl are optionally substituted with 1 to 3 R⁴;

each R⁴ is independently selected from the group consisting of H, C₁₋₆alkyl, C₂₋₆ alkenyl, and C₂₋₆ alkynyl, wherein said alkyl, alkenyl, andalkynyl are optionally substituted with one or more substituentsselected from the group consisting of halo, CN, NH₂, OH, and C₁₋₆alkoxy;

and n is 0-4.

In various embodiments, at least one pair of two R² together with thecarbon atom to which they are attached form a saturated or unsaturated3-8 membered cycloalkyl or heterocycloalkyl ring. In another embodiment,each pair of R² together with the carbon atom to which they are attachedform a saturated or unsaturated 3-8 membered cycloalkyl orheterocycloalkyl ring. In some cases, at least one pair of two R²together with the carbon atom to which they are attached form asaturated or unsaturated 5 membered cycloalkyl or heterocycloalkyl ring,wherein the heterocycloalkyl ring has 1 or 2 ring heteroatoms selectedfrom O, S, and N, and wherein said cycloalkyl and heterocycloalkyl ringare optionally substituted with 1 to 3 R³.

In various embodiments, at least one R2 or one pair of two R² togetherforming a ring comprise an α,β-unsaturated moiety. In one embodiment,one R² comprises an α,β-unsaturated moiety. In another embodiment, atleast one pair of two R² together forming a ring comprises anα,β-unsaturated moiety. In one embodiment, two pairs of two R² togetherforming a ring comprise α,β-unsaturated moieties.

In various embodiments, at least one pair of two R² together with thecarbon atom to which they are attached form a ring having the structure:

wherein * is the carbon atom to which each R² is attached, Z is O orNR⁴, and R⁵ is C₁₋₆alkyl, C₂₋₆alkenyl, C₆₋₁₀ aryl, or 3-7 memberedheterocycloalkyl ring having 1-3 ring heteroatoms selected from O, S,and N. In some embodiments, one pair of two R² together with the carbonatom to which they are attached form a ring having the structure:

In some embodiments, two pairs of two R² together with the carbon atomto which they are attached form a ring having the structure:

In various embodiments, at least one pair of two R² together with thecarbon atom to which they are attached form a ring having the structure:

In some embodiments, Z is O or NH. In one embodiment, Z is NH. Inanother embodiment, Z is O.

In some embodiments, at least one R² has a structure:

wherein W is O, S, or NR⁴; and Q is selected from the group consistingof C₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, N(R⁴)₂, and OR⁴. In someembodiments, W is O and Q is OR⁴. In some embodiments, R⁴ is H or C₁₋₆alkyl. In one embodiment, R⁴ is methyl.

The “L” comprises C₁₋₁₈alkylene or C₂₋₁₈alkenylene optionallyinterrupted with one or more of (i) non-adjacent heteroatom(s) selectedfrom O, S, and NR⁴, (ii) C(O)NR⁴, (iii) C₆₋₁₀ aryl, (iv) 5-10 memberedheteroaryl having 1-4 heteroatoms selected from N, O, and S, (v) 3-12membered cycloalkyl ring, and (vi) 3-7 membered heterocycloalkyl ringhaving 1-3 ring heteroatoms selected from O, S, and N, and said aryl,heteroaryl, cycloalkyl, and heterocycloalkyl are optionally substitutedwith one or more R⁴. In some cases, L is interrupted with 1-3 of anycombination of groups (i)-(vi). In some cases, L is interrupted with 1group of (i)-(vi). In some embodiments, L is uninterruptedC₁₋₁₈alkylene. In another embodiment, L is uninterrupted C₁₋₁₂alkylene.In various embodiments, L is C₁alkylene, C₂alkylene, C₃alkylene,C₄alkylene, C₅alkylene, C₆alkylene, C₇alkylene, C₈alkylene, C₉alkylene,C₁₀alkylene, C₁₁alkylene, or C₁₂alkylene. In one embodiment, L isC₇alkylene. In another embodiment, L is C₁₂alkylene.

In some embodiments, L is

In another embodiment, at least one of Y¹ and Y² is O. In anotherembodiment, each of Y¹ and Y² are O. In another embodiment, at least oneof Y¹ and Y² is NR⁴. In another embodiment, each of Y¹ and Y² are NR⁴.In some embodiments, R⁴ is H or C₁₋₆ alkyl. In one embodiment, R⁴ ismethyl.

In some embodiments, L is C₂₋₁₈alkenylene. In various embodiments, L isC₂alkenylene, C₃alkenylene, C₄alkenylene, C₅alkenylene, C₆alkenylene,C₇alkenylene, C₈alkenylene, C₉alkenylene, C₁₀alkenylene, C₁₁alkenylene,or C₁₂alkenylene. In some embodiments, L comprises one carbon-carbondouble bond. In some embodiments, L comprises two carbon-carbon doublebonds. In one embodiment, L is

and

indicates that the double bond is cis or trans. In another embodiment, Lis or

and

indicates that the double bond is cis or trans. In some embodiments, atleast one double bond is cis. In some embodiments, at least one doublebond is trans. In some embodiments, two double bonds are cis. In someembodiments, two double bonds are trans. In some embodiments, one doublebond is cis, and the other double bond is trans.

In some embodiments, L comprises a 3-12 membered cycloalkyl ringoptionally substituted with one or more R⁴. In some embodiments, Lcomprises a 5 or 6 membered cycloalkyl ring optionally substituted withone or more R⁴. In one embodiment, L is

In another embodiment, L is

In some embodiments, L is C₁₋₁₈alkylene or C₂₋₁₈alkenylene interruptedby at least one phenyl. In some embodiments, L is interrupted byC₁₋₁₈alkylene or C₂₋₁₈alkenylene interrupted by one phenyl. In someembodiments, L is interrupted by C₁₋₁₈alkylene or C₂₋₁₈alkenyleneinterrupted by two phenyls. In various embodiments, L is

In one embodiment, L is

In some embodiments, Y³ is O. In another embodiment, Y³ is NR⁴. In afurther embodiment, R⁴ is methyl.

In some embodiments, L is

In another embodiment, R⁴ is C₂₋₆ alkynyl. In another embodiment, R⁴ is4-butynyl.

Further provided are compounds as recited in Table A, or apharmaceutically acceptable salt thereof. Also provided are use ofcompounds recited in Table A, or a pharmaceutically acceptable saltthereof.

Specific compounds contemplated include those listed in Table 1, or apharmaceutically acceptable salt thereof:

TABLE 1 Compound # Structure 19

20

21

22

40-059

36-286

40-039

36-202

36-239

36-252

36-254

36-204

36-256

36-258

36-242

36-280

36-297

40-014

 36-252P

 36-252N

 P1

 P2

 P3

 P4

 P5

 P6

 P7

 P8

 P9

P10

P11

P12

P13

P14

P15

P16

25-4  

P17

P18

40-038

P19

P20

P21

P22

P23

P24

P25

P26

P27

P28

P29

The compounds disclosed herein can be in the form of a pharmaceuticallyacceptable salt. As used herein, the term “pharmaceutically acceptablesalt” refers to those salts which are, within the scope of sound medicaljudgment, suitable for use in contact with the tissues of humans andlower animals without undue toxicity, irritation, allergic response andthe like, and are commensurate with a reasonable benefit/risk ratio.Pharmaceutically acceptable salts are well known in the art. Forexample, S. M. Berge et al. describe pharmaceutically acceptable saltsin detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, which isincorporated herein by reference. Pharmaceutically acceptable salts ofthe compounds of this invention include those derived from suitableinorganic and organic acids and bases. Examples of pharmaceuticallyacceptable, nontoxic acid addition salts are salts of an amino groupformed with inorganic acids such as hydrochloric acid, hydrobromic acid,phosphoric acid, sulfuric acid and perchloric acid or with organic acidssuch as acetic acid, trifluoroacetic acid, oxalic acid, maleic acid,tartaric acid, citric acid, succinic acid or malonic acid or by usingother methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, glutamate, hemisulfate,heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate,lactobionate, lactate, laurate, lauryl sulfate, malate, maleate,malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate,oleate, oxalate, palmitate, pamoate, pectinate, persulfate,3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate,succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate,undecanoate, valerate salts, and the like. Salts of compounds containinga carboxylic acid or other acidic functional group can be prepared byreacting with a suitable base. Such salts include, but are not limitedto, alkali metal, alkaline earth metal, aluminum salts, ammonium,N⁺(C₁₋₄alkyl)₄ salts, and salts of organic bases such as trimethylamine,triethylamine, morpholine, pyridine, piperidine, picoline,dicyclohexylamine, N,N′-dibenzylethylenediamine, 2-hydroxyethylamine,bis-(2-hydroxyethyl)amine, tri-(2-hydroxyethyl)amine, procaine,dibenzylpiperidine, dehydroabietylamine, N,N′-bisdehydroabietylamine,glucamine, N-methylglucamine, collidine, quinine, quinoline, and basicamino acids such as lysine and arginine. This invention also envisionsthe quaternization of any basic nitrogen-containing groups of thecompounds disclosed herein. Water or oil-soluble or dispersible productsmay be obtained by such quaternization. Representative alkali oralkaline earth metal salts include sodium, lithium, potassium, calcium,magnesium, and the like. Further pharmaceutically acceptable saltsinclude, when appropriate, nontoxic ammonium, quaternary ammonium, andamine cations formed using counterions such as halide, hydroxide,carboxylate, sulfate, phosphate, nitrate, lower alkyl sulfonate and arylsulfonate.

Definitions

As used herein, the term “alkyl” refers to straight chained and branchedsaturated hydrocarbon groups containing one to thirty carbon atoms, forexample, one to twenty carbon atoms, or one to ten carbon atoms. Theterm C_(n) means the alkyl group has “n” carbon atoms. For example, C₄alkyl refers to an alkyl group that has 4 carbon atoms. C₁-C₇ alkylrefers to an alkyl group having a number of carbon atoms encompassingthe entire range (e.g., 1 to 7 carbon atoms), as well as all subgroups(e.g., 1-6, 2-7, 1-5, 3-6, 1, 2, 3, 4, 5, 6, and 7 carbon atoms).Nonlimiting examples of alkyl groups include, methyl, ethyl, n-propyl,isopropyl, n-butyl, sec-butyl (2-methylpropyl), t-butyl(1,1-dimethylethyl), 3,3-dimethylpentyl, and 2-ethylhexyl. Unlessotherwise indicated, an alkyl group can be an unsubstituted alkyl groupor a substituted alkyl group.

The term “alkylene” used herein refers to an alkyl group having asubstituent. For example, the term “alkylenehalo” refers to an alkylgroup substituted with a halo group. For example, an alkylene group canbe —CH₂CH₂— or —CH₂—. The term C_(n) means the alkylene group has “n”carbon atoms. For example, C₁₋₆ alkylene refers to an alkylene grouphaving a number of carbon atoms encompassing the entire range, as wellas all subgroups, as previously described for “alkyl” groups. Unlessotherwise indicated, an alkylene group can be an unsubstituted alkylenegroup or a substituted alkylene group.

The term “alkenyl” used herein refers to an unsaturated aliphatic groupanalogous in length and possible substitution to an alkyl groupdescribed above, but that contains at least one double bond. Forexample, the term “alkenyl” includes straight chain alkenyl groups(e.g., ethenyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl, octenyl,nonenyl, decenyl), and branched alkenyl groups. For example, a straightchain or branched alkenyl group can have six or fewer carbon atoms inits backbone (e.g., C₂-C₆ for straight chain, C₃-C₆ for branched chain).The term “C₂-C₆” includes chains having a number of carbon atomsencompassing the entire range (e.g., 2 to 6 carbon atoms), as well asall subgroups (e.g., 2-6, 2-5, 2-4, 3-6, 2, 3, 4, 5, and 6 carbonatoms). The term “C₃-C₆” includes chains having a number of carbon atomsencompassing the entire range (e.g., 3 to 6 carbon atoms), as well asall subgroups (e.g., 3-6, 3-5, 3-4, 3, 4, 5, and 6 carbon atoms). Unlessotherwise indicated, an alkenyl group can be an unsubstituted alkenylgroup or a substituted alkenyl group.

The term “alkenylene” used herein refers to an alkenyl group having asubstituent. For example, the term “alkenylenehalo” refers to an alkylgroup substituted with a halo group. For example, an alkylene group canbe —CH═CH—. The term C_(n) means the alkenylene group has “n” carbonatoms. For example, C₂₋₆alkenylene refers to an alkenylene group havinga number of carbon atoms encompassing the entire range, as well as allsubgroups, as previously described for “alkenyl” groups. Unlessotherwise indicated, an alkenylene group can be an unsubstitutedalkenylene group or a substituted alkenylene group.

As used herein, an alkylene or alkenylene which is “optionallyinterrupted” is understood to be an alkylene or alkenylene group inwhich at one or more (e.g., 1-5, 1-4, 1-3, 1-2, 1, 2, 3, 4, or 5)positions on the alkylene or alkenylene chain is inserted a groupselected from heteroatoms, aryl rings, heteroaryl rings, cycloalkylrings, or heterocycloalkyl rings. The interruptions can be consecutivefor various combinations of these interrupting groups (e.g., aheteroatom next to a heteroaryl moiety), except that two heteroatomscannot be adjacent or consecutive to each other. An alkylene oralkenylene in which no such inserted group is included is referred to as“uninterrupted”.

As used herein an alkylene or alkenylene which is optionally interruptedwith “one or more” groups is understood to be optionally interruptedwith from 1 to n−1 groups, wherein n is the number of carbon atoms inthe alkylene or alkenylene chain. For example, a C₆-alkylene which isoptionally interrupted with one or more groups can be interrupted withone, two, three, four, or five groups.

The term “alkynyl” used herein refers to an unsaturated aliphatic groupanalogous in length and possible substitution to an alkyl groupdescribed above, but that contains at least one triple bond. Forexample, the term “alkynyl” includes straight chain alkynyl groups(e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl, heptynyl, octynyl,nonynyl, decynyl), and branched alkynyl groups. For example, a straightchain or branched alkynyl group can have six or fewer carbon atoms inits backbone (e.g., C₂-C₆ for straight chain, C₄-C₆ for branched chain).The term “C₂-C₆” includes chains having a number of carbon atomsencompassing the entire range (e.g., 2 to 6 carbon atoms), as well asall subgroups (e.g., 2-6, 2-5, 2-4, 3-6, 2, 3, 4, 5, and 6 carbonatoms). The term “C₄-C₆” includes chains having a number of carbon atomsencompassing the entire range (e.g., 4 to 6 carbon atoms), as well asall subgroups (e.g., 4-6, 4-5, 4, 5, and 6 carbon atoms). Unlessotherwise indicated, an alkynyl group can be an unsubstituted alkynylgroup or a substituted alkynyl group.

As used herein, the term “cycloalkyl” refers to an aliphatic cyclichydrocarbon group containing three to twelve carbon atoms (e.g., 3, 4,5, 6, 7, 8, 9, 10, 11, or 12 carbon atoms). The term C_(n) means thecycloalkyl group has “n” carbon atoms. For example, C₅ cycloalkyl refersto a cycloalkyl group that has 5 carbon atoms in the ring. C₆-C₁₀cycloalkyl refers to cycloalkyl groups having a number of carbon atomsencompassing the entire range (e.g., 6 to 10 carbon atoms), as well asall subgroups (e.g., 6-7, 6-8, 7-8, 6-9, 6, 7, 8, 9, and 10 carbonatoms). Nonlimiting examples of cycloalkyl groups include cyclopropyl,cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Unlessotherwise indicated, a cycloalkyl group can be an unsubstitutedcycloalkyl group or a substituted cycloalkyl group. The cycloalkylgroups described herein can be isolated or fused to another cycloalkylgroup, a heterocycloalkyl group, an aryl group and/or a heteroarylgroup. When a cycloalkyl group is fused to another cycloalkyl group,then each of the cycloalkyl groups can contain three to twelve carbonatoms unless specified otherwise. Unless otherwise indicated, acycloalkyl group can be unsubstituted or substituted.

As used herein, the term “heterocycloalkyl” is defined similarly ascycloalkyl, except the ring contains one to three heteroatomsindependently selected from oxygen, nitrogen, and sulfur. In particular,the term “heterocycloalkyl” refers to a ring containing a total of threeto ten atoms (e.g., three to seven, or five to ten), of which 1, 2, 3 orthree of those atoms are heteroatoms independently selected from thegroup consisting of oxygen, nitrogen, and sulfur, and the remainingatoms in the ring are carbon atoms. Nonlimiting examples ofheterocycloalkyl groups include piperdine, pyrazolidine,tetrahydrofuran, tetrahydropyran, dihydrofuran, morpholine, and thelike.

Cycloalkyl and heterocycloalkyl groups can be saturated or partiallyunsaturated ring systems optionally substituted with, for example, oneto three groups, independently selected from C₁₋₆ alkyl, C₂₋₆ alkenyl,C₂₋₆ alkynyl, halo, CN, N(R⁴)₂, OR⁴, NO₂, oxo, ═S, ═NR⁴, CO₂R⁴, (C═O)R⁴,CON(R⁴)₂, C₆₋₁₀ aryl, and 5-10 membered heteroaryl. Heterocycloalkylgroups optionally can be further N-substituted with alkyl, alkylene-OH,alkylenearyl, and alkyleneheteroaryl. The heterocycloalkyl groupsdescribed herein can be isolated or fused to another heterocycloalkylgroup, a cycloalkyl group, an aryl group, and/or a heteroaryl group.When a heterocycloalkyl group is fused to another heterocycloalkylgroup, then each of the heterocycloalkyl groups can contain three to tentotal ring atoms, and one to three heteroatoms. Unless otherwiseindicated, a heterocycloalkyl group can be unsubstituted or substituted.

As used herein, the term “aryl” refers to a monocyclic aromatic group,such as phenyl. Unless otherwise indicated, an aryl group can beunsubstituted or substituted with one or more, and in particular one tofour groups independently selected from, for example, halo, alkyl,alkenyl, OCF₃, NO₂, CN, NC, OH, alkoxy, amino, CO₂H, CO₂alkyl, aryl, andheteroaryl. Other substituents are also contemplated, including C₀₋₃alkylene-halo, C₀₋₃ alkylene-CN, C₀₋₃ alkylene-NH₂, C₀₋₃ alkylene-OH,and C₀₋₃ alkylene-O—C₁₋₃alkyl. Aryl groups can be isolated (e.g.,phenyl) or fused to another aryl group (e.g., naphthyl, anthracenyl), acycloalkyl group (e.g. tetrahydronaphthyl), a heterocycloalkyl group,and/or a heteroaryl group. Exemplary aryl groups include, but are notlimited to, phenyl, chlorophenyl, methylphenyl, methoxyphenyl,trifluoromethylphenyl, nitrophenyl, 2,4-methoxychlorophenyl, and thelike. Throughout, the abbreviation “Ph” refers to phenyl and “Bn” refersto benzyl (i.e., CH₂phenyl).

As used herein, the term “heteroaryl” refers to a monocyclic aromaticring having 5 to 10 total ring atoms, and containing one to fourheteroatoms selected from nitrogen, oxygen, and sulfur atom in thearomatic ring. Unless otherwise indicated, a heteroaryl group can beunsubstituted or substituted with one or more, and in particular one tofour, substituents selected from, for example, halo, alkyl, alkenyl,OCF₃, NO₂, CN, NC, OH, alkoxy, amino, CO₂H, CO₂alkyl, aryl, andheteroaryl. Other substituents are also contemplated, including C₀₋₃alkylene-halo, C₀₋₃ alkylene-CN, C₀₋₃ alkylene-NH₂, C₀₋₃ alkylene-OH,and C₀₋₃ alkylene-O—C₁₋₃alkyl. In some cases, the heteroaryl group issubstituted with one or more of alkyl and alkoxy groups. Examples ofheteroaryl groups include, but are not limited to, thienyl, furyl,pyridyl, pyrrolyl, oxazolyl, triazinyl, triazolyl, isothiazolyl,isoxazolyl, imidazolyl, pyrazinyl, pyrimidinyl, thiazolyl, andthiadiazolyl.

As used herein, the term “alkoxy” or “alkoxyl” as used herein refers toa “—O-alkyl” group. The alkoxy or alkoxyl group can be unsubstituted orsubstituted.

As used herein, the term “substituted,” when used to modify a chemicalfunctional group, refers to the replacement of at least one hydrogenradical on the functional group with a substituent. Substituents caninclude, but are not limited to, alkyl, cycloalkyl, alkenyl,cycloalkenyl, alkynyl, heterocycloalkyl, aryl, heteroaryl, hydroxyl,oxy, alkoxy, heteroalkoxy, ester, thioester, carboxy, cyano, nitro,amino, amido, acetamide, and halo (e.g., fluoro, chloro, bromo, oriodo). When a chemical functional group includes more than onesubstituent, the substituents can be bound to the same carbon atom or totwo or more different carbon atoms.

As used herein, the phrase “optionally substituted” means unsubstituted(e.g., substituted with a H) or substituted. As used herein, the term“substituted” means that a hydrogen atom is removed and replaced by asubstituent. It is understood that substitution at a given atom islimited by valency. The use of a substituent (radical) prefix name suchas alkyl without the modifier “optionally substituted” or “substituted”is understood to mean that the particular substituent is unsubstituted.

As used herein, the term “α,β-unsaturated moiety” refers to a functionalgroup bearing an unsaturated bond between alpha and beta carbons (e.g.,an alkene) adjacent to a carbon having a double bond to a heteroatom,for example, oxygen (e.g., a carbonyl), sulfur (e.g., a thiocarbonyl),or nitrogen (e.g., an imine). An α,β-unsaturated moiety can be anelectrophile capable of undergoing an addition reaction, e.g., a Michaeladdition. Electrophilic α,β-unsaturated moieties which can undergo anaddition reaction are also called Michael acceptors. An α,β-unsaturatedmoiety can be part of a chain or a ring structure in a molecule. Anα,β-unsaturated moiety can have the structure

wherein W is a heteroatom (e.g., O, S, or N), and

indicates that the double bond can be cis or trans. Nonlimiting examplesof α,β-unsaturated moieties include α,β-unsaturated carbonyl compounds(e.g., enones, enals, α,β-unsaturated esters, and α,β-unsaturatedamides), α,β-unsaturated thioesters, α,β-unsaturated thiones,α,β-unsaturated thiolates, α,β-unsaturated imines, and α,β-unsaturatedamidines.

As used herein, the term “therapeutically effective amount” means anamount of a compound or combination of therapeutically active compounds(e.g., a NFκB modulator or combination of NFκB modulators) thatameliorates, attenuates or eliminates one or more symptoms of aparticular disease or condition (e.g., cancer), or prevents or delaysthe onset of one of more symptoms of a particular disease or condition.

As used herein, the terms “patient” and “subject” may be usedinterchangeably and mean animals, such as dogs, cats, cows, horses, andsheep (e.g., non-human animals) and humans. Particular patients orsubjects are mammals (e.g., humans). The terms patient and subjectinclude males and females.

As used herein, the term “pharmaceutically acceptable” means that thereferenced substance, such as a compound of the present disclosure, or aformulation containing the compound, or a particular excipient, are safeand suitable for administration to a patient or subject. The term“pharmaceutically acceptable excipient” refers to a medium that does notinterfere with the effectiveness of the biological activity of theactive ingredient(s) and is not toxic to the host to which it isadministered.

As used herein the terms “treating”, “treat” or “treatment” and the likeinclude preventative (e.g., prophylactic) and palliative treatment.

As used herein, the term “excipient” means any pharmaceuticallyacceptable additive, carrier, diluent, adjuvant, or other ingredient,other than the active pharmaceutical ingredient (API).

Synthesis of Compounds of the Disclosure

The compounds disclosed herein can be prepared in a variety of waysusing commercially available starting materials, compounds known in theliterature, or from readily prepared intermediates, by employingstandard synthetic methods and procedures either known to those skilledin the art, or in light of the teachings herein. Standard syntheticmethods and procedures for the preparation of organic molecules andfunctional group transformations and manipulations can be obtained fromthe relevant scientific literature or from standard textbooks in thefield. Although not limited to any one or several sources, classic textssuch as Smith, M. B., March, J., March's Advanced Organic Chemistry:Reactions, Mechanisms, and Structure, 5^(th) edition, John Wiley & Sons:New York, 2001; and Greene, T. W., Wuts, P. G. M., Protective Groups inOrganic Synthesis, 3^(rd) edition, John Wiley & Sons: New York, 1999,are useful and recognized reference textbooks of organic synthesis knownto those in the art. For example, the compounds disclosed herein can besynthesized by solid phase synthesis techniques including thosedescribed in Merrifield, J. Am. Chem. Soc. 1963; 85:2149; Davis et al.,Biochem. Intl. 1985; 10:394-414; Larsen et al., J. Am. Chem. Soc. 1993;115:6247; Smith et al., J. Peptide Protein Res. 1994; 44: 183; O'Donnellet al., J. Am. Chem. Soc. 1996; 118:6070; Stewart and Young, Solid PhasePeptide Synthesis, Freeman (1969); Finn et al., The Proteins, 3rd ed.,vol. 2, pp. 105-253 (1976); and Erickson et al., The Proteins, 3rd ed.,vol. 2, pp. 257-527 (1976). The following descriptions of syntheticmethods are designed to illustrate, but not to limit, general proceduresfor the preparation of compounds of the present disclosure.

The synthetic processes disclosed herein can tolerate a wide variety offunctional groups; therefore, various substituted starting materials canbe used. The processes generally provide the desired final compound ator near the end of the overall process, although it may be desirable incertain instances to further convert the compound to a pharmaceuticallyacceptable salt, ester or prodrug thereof.

In general, compounds of Formula I can be synthesized according toScheme 1.

Compounds having structure d can be synthesized using the procedureshown in Scheme 1. Condensation of an optionally substituted aniline awith appropriate reagents, e.g., chloral hydrate, ammonium hydroxide,and concentrated sulfuric acid, produces an isatin or isatin derivativecompound b. Reaction of b with an appropriate bifunctional compound L′produces dimers having structure c. Subsequent addition of anappropriate reagent R^(2′) gives compounds as described herein, i.e.,compounds of Formula I having structure d. Appropriate additionconditions will be known to those skilled in the art, but arecontemplated to include, without limitation, transition metal catalyzedadditions such as indium catalyzed additions (e.g., a Barbier reaction).

The addition reaction to form d can be catalyzed by appropriate reagentsselected based on the precise nature of compounds c and R^(2′). Forexample, when R^(2′) is an allyl halide compound, the coupling ofcompounds c and R^(2′) can be an indium-mediated allylation.Occasionally, the coupling reaction may not require a catalyst.

Compounds a, L′, and R^(2′) can be purchased commercially or prepared bya variety of methods from commercially-available starting materials. Forexample, L′ can be a dibromoallyl compound, such as dibromoheptane ordibromododecane. Similarly, R^(2′) can be an allyl halide compound, suchas methyl 2-(bromomethyl)acrylate.

Derivatization reactions to transform compounds having structure d intoother compounds of Formula I can be selected based on the nature of thesubstituent R² and the functionality desired. For example, R² cancomprise an ester group, which can be hydrolyzed (e.g., by treatmentwith tosic acid) to a carboxylic acid group, which can be furtherderivatized by methods known in the art to form a variety of functionalgroups, including by cyclization as shown in Scheme 2.

Additional compounds as described herein (i.e., spirocyclic compounds ofFormula I) having structure f or g can be synthesized using theprocedure shown in Scheme 2. For example, hydrolysis of optionallybis-methacrylate compound e (e.g., by treatment with tosic acid) leadsto an intramolecular cyclization, forming a compound having structure f.Reduction of compound e (e.g., via palladium-catalyzed hydrogenation)gives compounds having structure e′. Subsequent hydrolysis formsadditional compounds as described herein, i.e., compounds havingstructure g.

Additional synthetic procedures for preparing the compounds disclosedherein can be found in the Examples section.

Pharmaceutical Formulations, Dosing, and Routes of Administration

Further provided are pharmaceutical formulations comprising a compoundas described herein (e.g., compounds of Formula I or pharmaceuticallyacceptable salts of the compounds) and a pharmaceutically acceptableexcipient.

The compounds described herein can be administered to a subject in atherapeutically effective amount (e.g., in an amount sufficient toprevent or relieve the symptoms of a disorder associated with aberrantNFκB activity). The compounds can be administered alone or as part of apharmaceutically acceptable composition or formulation. In addition, thecompounds can be administered all at once, multiple times, or deliveredsubstantially uniformly over a period of time. It is also noted that thedose of the compound can be varied over time.

A particular administration regimen for a particular subject willdepend, in part, upon the compound, the amount of compound administered,the route of administration, and the cause and extent of any sideeffects. The amount of compound administered to a subject (e.g., amammal, such as a human) in accordance with the disclosure should besufficient to effect the desired response over a reasonable time frame.Dosage typically depends upon the route, timing, and frequency ofadministration. Accordingly, the clinician titers the dosage andmodifies the route of administration to obtain the optimal therapeuticeffect, and conventional range-finding techniques are known to those ofordinary skill in the art.

Purely by way of illustration, the method comprises administering, e.g.,from about 0.1 mg/kg up to about 100 mg/kg of compound or more,depending on the factors mentioned above. In other embodiments, thedosage ranges from 1 mg/kg up to about 100 mg/kg; or 5 mg/kg up to about100 mg/kg; or 10 mg/kg up to about 100 mg/kg. Some conditions requireprolonged treatment, which may or may not entail administering lowerdoses of compound over multiple administrations. If desired, a dose ofthe compound is administered as two, three, four, five, six or moresub-doses administered separately at appropriate intervals throughoutthe day, optionally, in unit dosage forms. The treatment period willdepend on the particular condition and type of pain, and may last oneday to several months.

Suitable methods of administering a physiologically-acceptablecomposition, such as a pharmaceutical composition comprising thecompounds disclosed herein (e.g., compounds of Formula I), are wellknown in the art. Although more than one route can be used to administera compound, a particular route can provide a more immediate and moreeffective reaction than another route. Depending on the circumstances, apharmaceutical composition comprising the compound is applied orinstilled into body cavities, absorbed through the skin or mucousmembranes, ingested, inhaled, and/or introduced into circulation. Forexample, in certain circumstances, it will be desirable to deliver apharmaceutical composition comprising the agent orally, throughinjection by intravenous, intraperitoneal, intracerebral(intra-parenchymal), intracerebroventricular, intramuscular,intra-ocular, intraarterial, intraportal, intralesional, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, urethral,vaginal, or rectal means, by sustained release systems, or byimplantation devices. If desired, the compound is administeredregionally via intrathecal administration, intracerebral(intra-parenchymal) administration, intracerebroventricularadministration, or intraarterial or intravenous administration feedingthe region of interest. Alternatively, the composition is administeredlocally via implantation of a membrane, sponge, or another appropriatematerial onto which the desired compound has been absorbed orencapsulated. Where an implantation device is used, the device is, inone aspect, implanted into any suitable tissue or organ, and delivery ofthe desired compound is, for example, via diffusion, timed-releasebolus, or continuous administration.

To facilitate administration, the compound is, in various aspects,formulated into a physiologically-acceptable composition comprising acarrier (e.g., vehicle, adjuvant, or diluent). The particular carrieremployed is limited only by chemico-physical considerations, such assolubility and lack of reactivity with the compound, and by the route ofadministration. Physiologically-acceptable carriers are well known inthe art. Illustrative pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (for example, see U.S. Pat. No. 5,466,468). Injectableformulations are further described in, e.g., Pharmaceutics and PharmacyPractice, J. B. Lippincott Co., Philadelphia. Pa., Banker and Chalmers,eds., pages 238-250 (1982), and ASHP Handbook on Injectable Drugs,Toissel, 4th ed., pages 622-630 (1986)). A pharmaceutical compositioncomprising the compound is, in one aspect, placed within containers,along with packaging material that provides instructions regarding theuse of such pharmaceutical compositions. Generally, such instructionsinclude a tangible expression describing the reagent concentration, aswell as, in certain embodiments, relative amounts of excipientingredients or diluents (e.g., water, saline or PBS) that may benecessary to reconstitute the pharmaceutical composition.

Compositions suitable for parenteral injection may comprisephysiologically acceptable sterile aqueous or nonaqueous solutions,dispersions, suspensions, or emulsions, and sterile powders forreconstitution into sterile injectable solutions or dispersions.Examples of suitable aqueous and nonaqueous carriers, diluents,solvents, or vehicles include water, ethanol, polyols (propylene glycol,polyethylene glycol, glycerol, and the like), suitable mixtures thereof,vegetable oils (such as olive oil) and injectable organic esters such asethyl oleate. Proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preserving,wetting, emulsifying, and dispersing agents. Microorganism contaminationcan be prevented by adding various antibacterial and antifungal agents,for example, parabens, chlorobutanol, phenol, sorbic acid, and the like.It may also be desirable to include isotonic agents, for example,sugars, sodium chloride, and the like. Prolonged absorption ofinjectable pharmaceutical compositions can be brought about by the useof agents delaying absorption, for example, aluminum monostearate andgelatin.

Solid dosage forms for oral administration include capsules, tablets,powders, and granules. In such solid dosage forms, the active compoundis admixed with at least one inert customary excipient (or carrier) suchas sodium citrate or dicalcium phosphate or (a) fillers or extenders, asfor example, starches, lactose, sucrose, mannitol, and silicic acid; (b)binders, as for example, carboxymethylcellulose, alginates, gelatin,polyvinylpyrrolidone, sucrose, and acacia; (c) humectants, as forexample, glycerol; (d) disintegrating agents, as for example, agar-agar,calcium carbonate, potato or tapioca starch, alginic acid, certaincomplex silicates, and sodium carbonate; (a) solution retarders, as forexample, paraffin; (f) absorption accelerators, as for example,quaternary ammonium compounds; (g) wetting agents, as for example, cetylalcohol and glycerol monostearate; (h) adsorbents, as for example,kaolin and bentonite; and (i) lubricants, as for example, talc, calciumstearate, magnesium stearate, solid polyethylene glycols, sodium laurylsulfate, or mixtures thereof. In the case of capsules, and tablets, thedosage forms may also comprise buffering agents. Solid compositions of asimilar type may also be used as fillers in soft and hard filled gelatincapsules using such excipients as lactose or milk sugar, as well as highmolecular weight polyethylene glycols, and the like.

Solid dosage forms such as tablets, dragees, capsules, pills, andgranules can be prepared with coatings and shells, such as entericcoatings and others well known in the art. The solid dosage forms mayalso contain opacifying agents. Further, the solid dosage forms may beembedding compositions, such that they release the active compound orcompounds in a certain part of the intestinal tract in a delayed manner.Examples of embedding compositions that can be used are polymericsubstances and waxes. The active compound can also be inmicro-encapsulated form, optionally with one or more excipients.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, solutions, suspensions, syrups, and elixirs. Inaddition to the active compounds, the liquid dosage form may containinert diluents commonly used in the art, such as water or othersolvents, solubilizing agents and emulsifiers, as for example, ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol,dimethylformamide, oils, in particular, cottonseed oil, groundnut oil,corn germ oil, olive oil, castor oil, and sesame seed oil, glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan, or mixtures of these substances, and the like.

Besides such inert diluents, the composition can also include adjuvants,such as wetting agents, emulsifying and suspending agents, sweetening,flavoring, and perfuming agents. Suspensions, in addition to the activecompound, may contain suspending agents, as for example, ethoxylatedisostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,microcrystalline cellulose, aluminum metahydroxide, bentonite,agar-agar, and tragacanth, or mixtures of these substances, and thelike.

Compositions for rectal administration are preferably suppositories,which can be prepared by mixing the compounds of the disclosure withsuitable non-irritating excipients or carriers such as cocoa butter,polyethylene glycol or a suppository wax, which are solid at ordinaryroom temperature, but liquid at body temperature, and therefore, melt inthe rectum or vaginal cavity and release the active component.

The compositions used in the methods of the invention may be formulatedin micelles or liposomes. Such formulations include stericallystabilized micelles or liposomes and sterically stabilized mixedmicelles or liposomes. Such formulations can facilitate intracellulardelivery, since lipid bilayers of liposomes and micelles are known tofuse with the plasma membrane of cells and deliver entrapped contentsinto the intracellular compartment.

Upon formulation, solutions will be administered in a manner compatiblewith the dosage formulation and in such amount as is therapeuticallyeffective. The formulations are easily administered in a variety ofdosage forms such as injectable solutions, drug release capsules and thelike. For parenteral administration in an aqueous solution, for example,the solution should be suitably buffered if necessary and the liquiddiluent first rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous and intraperitoneal administration.

The frequency of dosing will depend on the pharmacokinetic parameters ofthe agents and the routes of administration. The optimal pharmaceuticalformulation will be determined by one of skill in the art depending onthe route of administration and the desired dosage. See, for example,Remington's Pharmaceutical Sciences, 18th Ed. (1990) Mack PublishingCo., Easton, Pa., pages 1435-1712, incorporated herein by reference.Such formulations may influence the physical state, stability, rate ofin vivo release and rate of in vivo clearance of the administeredagents. Depending on the route of administration, a suitable dose may becalculated according to body weight, body surface areas or organ size.Further refinement of the calculations necessary to determine theappropriate treatment dose is routinely made by those of ordinary skillin the art without undue experimentation, especially in light of thedosage information and assays disclosed herein, as well as thepharmacokinetic data observed in animals or human clinical trials.

The precise dosage to be employed depends upon several factors includingthe host, whether in veterinary medicine or human medicine, the natureand severity of the condition, e.g., disease or disorder, being treated,the mode of administration and the particular active substance employed.The compounds may be administered by any conventional route, inparticular enterally, and, in one aspect, orally in the form of tabletsor capsules. Administered compounds can be in the free form orpharmaceutically acceptable salt form as appropriate, for use as apharmaceutical, particularly for use in the prophylactic or curativetreatment of a disease of interest. These measures will slow the rate ofprogress of the disease state and assist the body in reversing theprocess direction in a natural manner.

It will be appreciated that the pharmaceutical compositions andtreatment methods of the invention are useful in fields of humanmedicine and veterinary medicine. Thus the subject to be treated is inone aspect a mammal. In another aspect, the mammal is a human.

In jurisdictions that forbid the patenting of methods that are practicedon the human body, the meaning of “administering” of a composition to ahuman subject shall be restricted to prescribing a controlled substancethat a human subject will self-administer by any technique (e.g.,orally, inhalation, topical application, injection, insertion, etc.).The broadest reasonable interpretation that is consistent with laws orregulations defining patentable subject matter is intended. Injurisdictions that do not forbid the patenting of methods that arepracticed on the human body, the “administering” of compositionsincludes both methods practiced on the human body and also the foregoingactivities.

Methods of Use

The compounds described herein (e.g., the compounds of Formula I orcompounds of Table 1) can inhibit an NFκB pathway. In some embodiments,the compounds bind to NFκB, IκB α, IKK-beta, and IKK-alpha leading totheir inhibition and degradation, e.g., the compounds trigger or inhibitNFκB-mediated biological activity, such as gene expression. In variousembodiments, the compounds are NFκB modulators, e.g., the compoundschange, inhibit, or prevent one or more of the NFκB pathway's biologicalactivities.

The compounds disclosed herein are particularly advantageous for thetreatment of diseases or disorders caused by aberrant expression oractivity of an NFκB pathway. The incidence and/or intensity of diseasesor disorders associated with aberrant expression or activity of an NFκBpathway is reduced.

Transduction of the NFκB pathway signals are initiated through externalstimuli of a cell. A ligand, such as cytokines or TNFα, binds to itsplasma transmembrane cell receptor, activating the receptor. The signalis then transduced from the receptors through the canonical NFκB pathway(involving NFκB1) and/or the alternative NFκB pathway (involving NFκB2).In the canonical pathway, a complex of IκBα kinase (IKK) proteins,IKKα/β/γ, is activated and phosphorylates the protein IκBα, which isassociated with p50 and p65, causing dissociation of IκBα. The IκBαinteraction with p50 and p65 is inhibitory, and p50/p65 is thenactivated through dissociation of IκBα. The p50/p65 complex is importedinto the nucleus where it acts as a transcription factor, thusactivating gene transcription. In the alternative pathway, the signalfrom the cell receptor is transmitted through the protein NIK, whichactivates IKKα. This ultimately activates the p52/ReIB complex. Thep52/ReIB complex is imported into the nucleus where it also acts as atranscription factor and activates gene transcription.

Increased expression and/or activity of an NFκB pathway includesoverexpression or hyperactivity of any component of an NFκB pathway.Overexpression and/or hyperactivity of the NFκB pathways is well knownto cause many adverse conditions. These include, for example, cancer,autoimmune diseases, inflammatory diseases, diabetes, cardiovasculardiseases, and neurological diseases. Cancer includes but is not limitedto ovarian cancer, breast cancer, prostate cancer, colon cancer, livercancer, brain cancer, kidney cancer, lung cancer, leukemia, lymphoma,multiple myeloma, thyroid cancer, bone cancer, esophageal cancer, andpancreatic cancer. Inflammatory diseases include but are not limited toarthritis, rheumatoid arthritis, atherosclerosis, multiple sclerosis,asthma, inflammatory bowel disease, Crohn's disease, gastritis,pancreatitis, systemic inflammatory response syndrome, and chronicinflammatory demyelinating polyradiculoneuritis.

NFκB selective inhibitors can be used for cancer prevention andtreatment. The relationship between NFκB activation andinflammation-associated cancer have been demonstrated using severalmouse models. NFκB activation has been implicated in inflammationassociated liver, prostate and colon cancer induction in humans andmouse models. Several antioxidants having electrophilic capacity such ascycloheptenone prostaglandins, dimethylsulfoxide, glutathione andnon-steroidal anti-inflammatory drugs (NSAIDs) Ibuprofen, sulindac, aswell as curcumin inhibit NFκB activity but do not show high selectivity.Aspirin, sulfasalazine, SC-514, and PS-1145 also inhibit NFκB byinterrupting phosphorylation of IKK.

Compounds of Formula I display high selectivity for growth inhibitionand/or induction of apoptosis in cancer cells, e.g., in ovarian cancercells.

The disclosed methods include methods for treating disease or disordercapable of being modulated by inhibition of the NFκB pathway, e.g.,cancer, comprising administering to a subject a compound that binds acomponent of the NFκB pathway. In some examples, the compound disruptsbinding of a protein which activates the NFκB pathway. In one example,the method includes use of a compound that disrupts binding of a proteinto TNFα. In another example, the method includes use of a compound thatdisrupts binding of a protein to IKKβ. In another example, the compoundprevents translocation of NFκB to the nucleus.

Provided herein is a method of modulating the NFκB pathway in a cell,comprising contacting the cell with a compound or a composition asdisclosed herein (e.g., the compounds of Formula I or as shown in TableI) in an amount sufficient to modulate the NFκB pathway. The contactingof the cell can occur in vitro or in vivo. In some cases, contacting ofthe cell occurs in vitro. In other cases, contacting of the cell occursin vivo. Therefore, the disclosure includes administering one or more ofa compound described herein to a subject, such as a human, in needthereof. In some embodiments, the subject suffers from a disease ordisorder associated with aberrant activity of the NFκB pathway.Disorders associated with aberrant activity of the NFκB pathway include,but are not limited to, cancer (e.g., ovarian cancer), autoimmunediseases, inflammatory diseases, diabetes, cardiovascular diseases, andneurological diseases. Specifically contemplated cancers include ovariancancer, breast cancer, prostate cancer, colon cancer, liver cancer,brain cancer, kidney cancer, lung cancer, leukemia, lymphoma, multiplemyeloma, thyroid cancer, bone cancer, esophageal cancer, and pancreaticcancer.

The disclosed methods utilize compounds that inhibit the NFκB pathway,for treating, e.g., cancer. Methods for assessing the usefulness of acompound for treating cancer are known to those of skill in the art. Forexample, compounds may be assessed using models of cancer, includingcells (such as ovarian cancer cells), animal models (such as mousexenograph or other cancer models), or in human subjects having, e.g.,ovarian cancer.

The compounds described herein can be used to decrease or prevent cancerin human subjects with e.g., ovarian cancer. In a particular example, acompound or mixture is administered orally, such as by mixing withdistilled water. In another example, a test compound or mixture isadministered intravenously, such as in saline or distilled water. Insome examples, treatment with test compound may be a single dose orrepeated doses. The test compound may be administered about every 6hours, about every 12 hours, about every 24 hours (daily), about every48 hours, about every 72 hours, or about weekly. Treatment with repeateddoses may continue for a period of time, for example for about 1 week to12 months, such as about 1 week to about 6 months, or about 2 weeks toabout 3 months, or about 1 to 2 months. Administration of a compound mayalso continue indefinitely. Doses of test compound are from about 0.1mg/kg to about 400 mg/kg, such as about 1 mg/kg to about 300 mg/kg,about 2 mg/kg to 200 mg/kg, about 10 mg/kg to about 100 mg/kg, about 20mg/kg to about 75 mg/kg, or about 25 mg/kg to about 50 mg/kg.

It will be understood that the methods and compositions described hereinfor treating cancer, comprising administering a compound that inhibitsthe NFκB pathway, are applicable to methods of treating other diseasesrelated to NFκB activity, such as those described above. The methods forassessing the effectiveness of test compounds for treating such diseasesin cells, appropriate animal models, or affected subjects are known toone of skill in the art.

Uses of the compounds disclosed herein in the preparation of amedicament for treating diseases or disorders related to NFκB activityalso are provided herein.

The disclosure herein will be understood more readily by reference tothe following examples, below.

EXAMPLES

The following examples are provided for illustration and are notintended to limit the scope of the disclosure.

Synthetic Procedures for Compounds of Formula I

General Experimental Procedures. All reagents were purchased fromcommercial sources and were used without further purification. Flashchromatography was carried out on silica gel (200-400 mesh). Thin layerchromatography (TLC) was performed on pre-coated EMD silica gel 60 F254plates and observed under UV light at 254 nm and analyzed with basicpotassium permanganate staining. Column chromatography was performedwith silica gel (230-400 mesh, grade 60, Fisher scientific, USA). ¹H NMR(500 MHz) and ¹³C NMR (125 MHz) spectra were recorded in chloroform-d orDMSO-d6 on a Varian-500, Varian-600 and Bruker-500 spectrometer (DMSO-d62.50 ppm for 1H and 39.00 ppm for 13C and CDCl3 was 7.26 ppm for 1H and77.00 ppm for 13C. Proton and carbon chemical shifts were reported inppm relative to the signal from residual solvent proton and carbon. Thepurity of all final compounds was ≥95% as determined by analytical HPLCon a reverse-phase column (Zorbax 300SB C18, 2.1×150 mm, 5 μm particlesize) using an Agilent 1200 series system with UV detector (214 nm and254 nm) using a binary water/acetonitrile containing system 0.1%trifluoracetic acid (TFA) as eluent. Analytical HPLC was carried out ona 250×4.60 mm C-18 column using gradient conditions (10-100% B, flowrate=1.0 mL/min, 15 min). The eluents used were: solvent A (H₂O with0.1% Formic acid) and solvent B (CH₃CN with 0.1% formic acid).

Example 1: Compounds of Formula (I) Having an Alkylene Linker

Step i

Isatin (1 equivalent) and dry DMF were combined in a dry round bottomflask. The reaction mixture was cooled to 0° C., and then NaH (0.45equivalent) was added. The reaction mixture was stirred for 15 minutes,and then 1,n-dibromoalkene (0.45 equivalent) was added. The reaction waswarmed to room temperature and stirred for 24 hours. The crude mixturewas diluted in ethyl acetate and washed with ammonium chloride andbrine. The organic layer was separated and dried with magnesium sulfate,filtered, and then evaporated to give the pure intermediate compound.

Step ii

In a dry round bottom flask was dissolved isatin (1 equivalent) inTHF:water (3:2), followed by addition of indium metal powder (2.2equivalents). The reaction mixture was stirred for 10 minutes, and thenmethyl 2-(bromomethyl) acrylate (2.2 equivalents) was added. Thereaction mixture was stirred for 24 hours and the progress of thereaction was monitored by thin layer chromatography using 70% ethylacetate in hexane solvent. Following reaction completion, the reactionmixture was diluted with ethyl acetate and washed with 0.1% HCl,followed by brine, and dried over magnesium sulfate. The crude mixturewas purified by column chromatography using a hexane and ethyl acetategradient to obtain the desired acyclic intermediate compound.

Step iii

In a round bottom flask was dissolved the acyclic intermediate compound(1 equivalent) in dry dichloromethane. The reaction mixture was cooledto 0° C. followed by addition of p-toluenesulfonic acid monohydrate salt(2.2 equivalents). The reaction mixture was stirred at room temperaturefor 12 hours under inert atmosphere. Completion of the reaction wasmonitored by thin layer chromatography. The crude mixture was washedwith brine and extracted using dichloromethane, dried over magnesiumsulfate, and purified via column chromatography using hexane and ethylacetate gradient to obtain the desired product.

Example 2:1′,1′″-(heptane-1,7-diyl)bis(4-methyl-3,4-dihydro-5H-spiro[furan-2,3′-indoline]-2′,5-dione)

Step i: Dimethyl2,2′-((heptane-1,7-diylbis(3-hydroxy-2-oxoindoline-1,3-diyl))bis(methylene))diacrylate

To dimethyl2,2′-((heptane-1,7-diylbis(3-hydroxy-2-oxoindoline-1,3-diyl))bis(methylene))diacrylate(1 equivalent) in a round bottom flask was added dry THF via syringe. Tothe reaction mixture was added Pd/C (5% by weight on activated carbon),and nitrogen gas was bubbled though the reaction mixture for about 10minutes. The reaction mixture was gently vacuumed and was kept underhydrogen atmosphere for 24 hours. After completion, the mixture waspassed through a bed of celite and purified by column chromatographyusing hexane and ethyl acetate gradient to obtain the product, dimethyl2,2′-((heptane-1,7-diylbis(3-hydroxy-2-oxoindoline-1,3-diyl))bis(methylene))diacrylate.

Step ii:1′,1′″-(heptane-1,7-diyl)bis(4-methyl-3,4-dihydro-5H-spiro[furan-2,3′-indoline]-2′,5-dione)

A round bottom flask was charged with dimethyl2,2′-((heptane-1,7-diylbis(3-hydroxy-2-oxoindoline-1,3-diyl))bis(methylene))diacrylate (1 equivalent) and dry dichloromethane. The reaction mixturewas cooled to 0° C. followed by addition of p-toluenesulfonic acidmonohydrate salt (1.2 equivalents). The reaction mixture was stirred atroom temperature for 12 hours under inert atmosphere. Completion of thereaction was monitored by thin layer chromatography. The crude mixturewas washed with brine and extracted using dichloromethane, dried overmagnesium sulfate, and purified via column chromatography using hexaneand ethyl acetate gradient to yield the title compound.

Example 3:1,1″-(octane-1,8-diyl)bis(4,7-dimethyl-4′-methylenespiro[indoline-3,2′-pyrrolidine]-2,5′-dione)

Step i: 1,1′-(heptane-1,7-diyl)bis(4,7-dimethylindoline-2,3-dione)

In a dry round bottom flask were combined 4,7-dimethylindoline-2,3-dione(1 equivalent) and dry DMF. The reaction mixture was cooled to 0° C.,followed by addition of NaH (0.45 equivalent). The reaction mixture wasstirred for 15 minutes followed by addition of 1,3-dibromoalkene (0.45equivalents). The reaction was warmed to room temperature and stirred atroom temperature for 24 hours. The crude mixture was diluted in ethylacetate and washed with ammonium chloride followed by brine. The organiclayer was separated and dried with magnesium sulfate, filtered, andevaporated to yield the desired product.

Step ii: dimethyl2,2′-((heptane-1,7-diylbis(3-hydroxy-4,7-dimethyl-2-oxoindoline-1,3-diyl))bis(methylene))diacrylate

In a dry round bottom flask were combined1,1′-(heptane-1,7-diyl)bis(4,7-dimethylindoline-2,3-dione) (1equivalent) and THF:water (3:2), followed by addition of indium metalpowder (2.2 equivalents). The reaction mixture was stirred for 10minutes followed by addition of methyl 2-(bromomethyl) acrylate (2.2equivalents). The reaction mixture was stirred for 24 hours and theprogress of the reaction was monitored by thin layer chromatographyusing 70% ethyl acetate in hexane solvent. Following reactioncompletion, the reaction mixture was diluted with ethyl acetate andwashed with 0.1% HCl, followed by brine, and dried using magnesiumsulfate. The crude mixture was purified by column chromatography using ahexane and ethyl acetate gradient to yield the desired product.

Step iii:1,1″-(heptane-1,7-diyl)bis(4,7-dimethyl-4′-methylenespiro[indoline-3,2′-pyrrolidine]-2,5′-dione)

Dimethyl2,2′-((heptane-1,7-diylbis(3-hydroxy-4,7-dimethyl-2-oxoindoline-1,3-diyl))bis(methylene))diacrylate(1 equivalent) was dissolved in dry dichloromethane in a round bottomflask. The reaction mixture was cooled to 0° C., followed by addition ofp-toluenesulfonic acid monohydrate salt (2.2 equivalents). The reactionmixture was stirred at room temperature for 12 hours under inertatmosphere. Completion of the reaction was monitored by thin layerchromatography. The crude mixture was washed with brine and extractedusing dichloromethane, dried over magnesium sulfate, and purified bycolumn chromatography using a hexane and ethyl acetate gradient to yieldthe title compound.

Example 4:1,1″-(1,4-phenylenebis(methylene))bis(4′-methylenespiro[indoline-3,2′-pyrrolidine]-2,5′-dione)

Step i: 1,1′-(1,4-phenylenebis(methylene))bis(indoline-2,3-dione)

In a dry round bottom flask were combined isatin (1 equivalent) and dryDMF. The reaction mixture was cooled to 0° C. followed by addition ofNaH (0.45 equivalents). The reaction mixture was stirred for 15 minutesfollowed by addition of 1,4-bis(bromomethyl)benzene (0.45 equivalents).The reaction was warmed to room temperature and stirred for 24 hours.The crude mixture was diluted in ethyl acetate and washed with ammoniumchloride, followed by brine. The organic layer was separated and driedwith magnesium sulfate, filtered, and evaporated to give the desiredcompound.

Step ii: dimethyl2,2′-(((1,4-phenylenebis(methylene))bis(3-hydroxy-2-oxoindoline-1,3-diyl))bis(methylene))diacrylate

In a dry round bottom flask were combined1,1′-(1,4-phenylenebis(methylene))bis(indoline-2,3-dione) (1 equivalent)and THF:water (3:2), followed by addition of indium metal powder (2.2equivalents). The reaction mixture was stirred for 10 minutes followedby addition of methyl 2-(bromomethyl) acrylate (2.2 equivalents). Thereaction mixture was stirred for 24 hours and progress of the reactionwas monitored by thin layer chromatography using 70% ethyl acetate inhexane solvent. Following completion of the reaction, the mixture wasdiluted with ethyl acetate and washed with 0.1% HCl, followed by brine,and dried over magnesium sulfate. The crude mixture was purified bycolumn chromatography using a hexane and ethyl acetate gradient to yieldthe desired compound.

Step ii:1,1″-(1,4-phenylenebis(methylene))bis(4′-methylenespiro[indoline-3,2′-pyrrolidine]-2,5′-dione)

Dimethyl2,2′-(((1,4-phenylenebis(methylene))bis(3-hydroxy-2-oxoindoline-1,3-diyl))bis(methylene))diacrylate(1 equivalent) was dissolved in dry dichloromethane in a round bottomflask. The reaction mixture was cooled to 0° C. followed by addition ofp-toluenesulfonic acid monohydrate salt (2.2 equivalents). The reactionmixture was stirred at room temperature for 12 hours under inertatmosphere. Completion of the reaction was monitored by thin layerchromatography. The crude mixture was washed with brine and extractedusing dichloromethane, dried over magnesium sulfate, and purified bycolumn chromatography using a hexane and ethyl acetate gradient to yieldthe title compound.

Biological Assay Data Example 5: Cell Viability Assay

MiaPaCa2, A2780, and OVCAR5 cells were plated at 4000 cells/well in a96-well plate and allowed to adhere overnight. The following day, cellswere treated with compounds using ˜10-fold dilutions starting at 1000nM. PrestoBlue reagent (Invitrogen #A13262) was added to cells after 72hour drug incubation to assess the growth inhibition. Fluorescenceexcitation/emission was measured at 560/590 nM using a SpectraMax M5^(e)instrument. Growth inhibition was calculated using100−[100*(Samples−T₀)/(T₁₀₀−T₀)]. T₀ is the vehicle control readingimmediately following drug addition and T₁₀₀ is the control reading atthe end of 72 hour incubation. Results are presented in Table 2, below.

TABLE 2 IC₅₀ (μM) Compound # A2780 OVCAR5 MiaPaCa2 25-4  0.99 ± 0.113.13 ± 0.55 6.08 40-059 0.42 ± 0.04 0.32 ± 0.02 36-286 0.23 ± 0.02 0.09± 0.01 40-038 0.40 ± 0.06 0.20 ± 0.04 36-202 0.29 ± 0.04 0.09 ± 0.010.91 36-239 0.70 ± 0.09 0.30 ± 0.02 1.05 36-252 0.32 0.32 0.76 36-2540.60 ± 0.07 0.32 1.70 36-204 1.08 ± 0.01 0.48 ± 0.06 2.16 36-256 0.49 ±0.06 0.41 ± 0.06 1.47 36-258 0.77 ± 0.14 0.83 ± 0.12 36-242 0.50 ± 0.060.47 ± 1.29 1.94 40-014 0.31 ± 0.02 0.18 ± 0.01 36-280 0.35 ± 0.05 0.37± 0.04 36-297 Inactive Inactive

Example 6: Cell-Based Growth Inhibition and Apoptosis Assays

The in vitro activity of the dimers was assessed through cell-basedassays to determine their ability to selectively inhibit growth andinduce apoptosis in ovarian cancer cells. In a 3-day growth assay,compound 36-242 was shown to be 10-fold more potent than monomericcompound 19. In the same assay, reduced compound 36-297.

In a panel of high grade serous carcinoma (HGSC) ovarian cancer celllines, the dimer with the 7-carbon linker 36-252 (sub μM) was morepotent than analog 19 and a dimer with a 12-carbon linker 36-242. TheIC₅₀ value of all three inhibitors was greater than 20 μM in this cellline. The average IC₅₀ value of 36-252 in HGSC cell lines was 0.78 μM,demonstrating that 36-252 inhibits growth of HGSC (efficacy) withgreater than 25-fold selectivity over nontransformed cell line(toxicity), corresponding to an apparent therapeutic index of >25.

Monomeric compound 19, 36-252, and 36-242 were evaluated innon-transformed HGSC precursor fallopian tube epithelial (FTE) cellsFT282. Unlike HGSC cell lines the IC₅₀ values of the inhibitors in theFT282 cells were greater than 20 μM, which is 25-fold better than theaverage IC₅₀ value (0.78 μM) of 36-252 in HGSC cell lines.

Example 7: Caspase and kB-Luciferase Assays

To determine the effect of 36-252 on apoptosis, a small panel of HGSCcell lines was treated with 1 μM 36-252 for 6 hours (1 μM offlavopiridol was used as a positive control). Caspases are a class ofcysteine proteinases that are activated during apoptosis and measuringcaspase activity is routinely used to detect activation of apoptoticsignaling. Activation of caspase 3/7 was assessed using a luciferaseassay. Increased caspase 3/7 activity was seen in three of the five HGSCcell lines.

2500 cells were plated in a 384-well black walled clear bottom plate at2,500 cells per well in 100 μL per well in the presence of 4.5 μMdoxycycline. Cells were incubated overnight to adhere to plate. Cellswere then treated at concentrations indicated in FIG. 1. After cellswere treated and plate was allowed to incubate, Caspase-glo (PromegaG8093) reagent was added and the plate was allowed to incubate for 30min. The plate was then read for luminescence at 1000 ms. Presto-bluewas added at a volume of 1/10 total volume per well (2.5 μL) and cellswere allowed to incubate for 10 min. The plate was then read forfluorescence at 560_(ex)/590em. Values were calculated by:([Luminescence*100]/Fluorescence)/DMSO_(avg).

A549 luciferase cells were seeded in white 96-well plates at a densityof 50,000 cells per well and incubated overnight. Cells were thentreated with the compound concentrations indicated in FIG. 3 for 2hours. Cells were stimulated with 20 ng/mL TNFα (Panomics) for 3 hours.AlamarBlue (abSerotec) was added (which served as a control for seedingand viability) and the cells were incubated for 3 additional hours.ONE-Glo Luciferase reagent (Promega) was added to each well and cellswere incubated at room temperature for an additional 10 minutes beforeluminescence was measured at 1000 ms integration on a SpectraMax M5eplate reader.

The effect 36-252 and 36-297 on NFκB driven gene expression wasevaluated using a cancer cell line specifically designed to monitor theactivity of NFκB in response to TNF-α. Cells were seeded onto 96-wellplates and allowed to attach overnight. The cells were treated with36-252 and 36-297 (20, 4, and 0.8 μM) for 2 hours, followed by TNF-β foran additional 6 hours. Under multiplexing conditions the cells wereassayed for viability using Alamar Blue (Ab Serotec) and the NFκBtranscription activity using the ONE-Glo luciferase system (Promega).Dose-dependent inhibition was observed with 36-252 and not 36-297, whichdemonstrates the need for the Michael acceptor in 36-252 to inhibit NFκBmediated gene expression.

Example 8: Western Blot Analyses

Crosslinking of proteins in the IKK complex was studied as follows.A2780 cells were treated with 10 μM of analog 19 or 36-252 and incubatedfor 2 hours. High molecular weight bands in the NFκB and IκBα blots wereobserved, indicating that crosslinking had occurred. A time course studywith 36-252 showed a band between 150 and 250 kDa in addition to theIκBα-NFκB bands, corresponding to a possible IκBα-NFκB-IKKα/β complexresulting in degradation of IKK α/β and PARP cleavage.

Cells were washed with cold 1×PBS 3 times and scraped before being lysedby a buffer containing the following: 50 mM Tris, 100 mM NaCl, 1% NP-40,2 nM EDTA, 20% SDS, 20×PPI (Na₃VO₄, NAF, β-glycerophosphate) and 1mmol/L PMSF. After collection, samples were incubated on ice for 30minutes and vortexed in 15 minute intervals. Samples were thencentrifuged at 14,000 rpm for 10 minutes at 4° C. and supernatant wascollected. Protein quantification was determined by BCA Protein Assay(Pierce #23225). 20-40 ug protein samples were run on 4-15% gradientgels (BioRad) in 1×TRIS-Glycine-SDS Buffer (Research ProductsInternational Corporation #T32080) at 120V for ˜60 minutes and separatedby SDS-page electrophoresis. Samples were transferred to a PVDF membraneby semi-dry transfer method (ThermoScientific #35035) run at 18V for ˜35minutes. Membranes were blocked in 5% milk diluted in 1×-Tris BufferedSaline with 0.1% Tween (1×TBST) for 1 hour at room temperature rockingat low speed. Primary antibodies were diluted in 5% milk in 1×TBST andwere rocked gently overnight in 4° C. Membranes were incubated with theappropriate HRP-conjugated secondary antibody for 1 hour at roomtemperature while gently rocking. 3 washes (10 minute) with 1×TBSToccurred before and after secondary antibody. ECL Prime (GE Healthcare#RPB2236) was used to detect protein expression

Example 9: Side Population Analysis

Cancer cells with the ability to efflux Hoechst 33342 dye are termed“side population” (SP). SP cells are far more tumorigenic because theycontain a sub-population of cells with cancer stem-like properties Amajority of ovarian cancer patients relapse and become drug resistantwhich has been attributed to the inability to kill stem cells enrichedin the SP. The effects of 36-252 and Verapamil (positive control) on theside population in ovarian cancer cell lines (A2780 and OVCAR5) byanalyzing the fluorescent profile using flow cytometry methods. WhileVerapamil eliminated about 60% of SP cells in both cell lines, 36-252eliminated >85% of OVCAR5 SP cells and >98% of A2780 SP cells.

2 million OVCAR-5 or A2780 cells were treated with 60 μM of verapamil,or 36-252 and vehicle control for 15 min at 37° C. Hoechst 33342 dye (5μg) was added to each tube and incubated for 45 min at 37° C. Tubes werevortexed slightly after every 5 min. Cells were resuspended in a singlecell suspension after centrifugation and kept on ice before acquisition.The side-population was measured by flow cytometry.

Example 10: Dose Limiting Toxicities

Six to eight-week-old C57 Albino mice (18-22 grams) were used in thisstudy and were randomly assigned. For MTD studies, 3 mice were randomlyassigned to six groups. Animals (Group 1-6) received a single dose of(0, 1, 5, 10, 20 and 100 mg/Kg in 30 mL neat DMSO) of 36-252 viaintraperitoneal injection. Mice were sacrificed after 48 hours, andblood and serum samples were subjected to hematology and clinicalchemistry analyses. For group 7, a 6-day, 5 mg/kg/day study was carriedout followed by submission of samples for hematology and clinicalchemistry analyses. Cohort summaries are presented in Table 3, below.

TABLE 3 Dose No. of Treated/ Grp (mg/Kg) doses Alive 1 Vehicle 1 (3/3) 2 1 1 (3/3) 3  5 1 (3/3) 4  10 1 (3/3) 5  20 1 (3/3) 6 100 1 (3/3) 7Vehicle 7 (3/3) 8  5 7 (3/3)

In the phase I study, the lethal dose or maximum tolerated dose of36-252 was evaluated. Even at 100 mg/kg, no sign of overt toxicity wasobserved. In the phase II study, three mice at 5 mg/kg/day wereadministered intraperitoneally for six days. No mortality or abnormalclinical levels were observed, nor was any statistical difference inmean body weight observed. Compared to DMSO treated mice, hematologicalanalysis showed no significant differences in relative WBC, RBC, orhemoglobin numbers in 36-252 treated mice.

In view of the many possible embodiments to which the principles of thedisclosure may be applied, it should be recognized that the illustratedembodiments are only examples and should not be taken as limiting thescope of the invention.

1. A compound, or pharmaceutically acceptable salt thereof, having thestructure of Formula I:

wherein X is O, S, or NR⁴; L is C₁₋₁₈alkylene or C₂₋₁₈alkenyleneoptionally interrupted with one or more of (i) non-adjacentheteroatom(s) selected from O, S, and NR⁴, (ii) C(O)NR⁴, (iii) C₆₋₁₀aryl, (iv) 5-10 membered heteroaryl having 1-4 heteroatoms selected fromN, O, and S, (v) 3-12 membered cycloalkyl ring, and (vi) 3-7 memberedheterocycloalkyl ring having 1-3 ring heteroatoms selected from O, S,and N, and said aryl, heteroaryl, cycloalkyl, and heterocycloalkyl areoptionally substituted with one or more R⁴; each R¹ is independentlyselected from the group consisting of C₁₋₆ alkyl, halo, CN, N(R⁴)₂, OR⁴,NO₂, CO₂R⁴, (C═O)R⁴, CON(R⁴)₂, NR⁴(C═O)R⁵, C₆₋₁₀ aryl, and 5-10 memberedheteroaryl having 1-4 heteroatoms selected from N, O, and S, and saidalkyl can be optionally substituted with 1 to 3 R³; each R² isindependently selected from the group consisting of C₁₋₆ alkyl, C₂₋₆alkenyl, C₂₋₆ alkynyl, halo, OR⁴, COR⁴, CO₂R⁴, CON(R⁴)₂, N(R⁴)₂, andSR⁴, or a pair of two R² together with the carbon atom to which they areattached form a saturated or unsaturated 4-8 membered cycloalkyl orheterocycloalkyl ring, wherein the heterocycloalkyl ring has 1 or 2 ringheteroatoms selected from O, S, and N, and wherein said alkyl, alkenyl,alkynyl, cycloalkyl ring, and heterocycloalkyl ring are optionallysubstituted with 1 to 3 R³, with the proviso that at least one R² or onepair of two R² together forming a ring comprise an α,β-unsaturatedmoiety; each R³ is independently selected from the group consisting ofC₁₋₆ alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, halo, CN, N(R⁴)₂, OR⁴, NO₂, oxo,═S, ═NR⁴, CO₂R⁴, (C═O)R⁴, CON(R⁴)₂, C₆₋₁₀ aryl, and 5-10 memberedheteroaryl having 1-4 ring heteroatoms selected from N, O, and S, andwherein said alkyl, alkenyl, and alkynyl are optionally substituted with1 to 3 R⁴; each R⁴ is independently selected from the group consistingof H, C₁₋₆ alkyl, C₂₋₆ alkenyl, and C₂₋₆ alkynyl, wherein said alkyl,alkenyl, and alkynyl are optionally substituted with one or moresubstituents selected from the group consisting of halo, CN, NH₂, OH,and C₁₋₆ alkoxy; and n is 0-4.
 2. The compound or salt of claim 1,wherein at least one pair of two R² together with the carbon atom towhich they are attached form a saturated or unsaturated 3-8 memberedcycloalkyl or heterocycloalkyl ring.
 3. The compound or salt of claim 2,wherein each pair of R² together with the carbon atom to which they areattached form a saturated or unsaturated 3-8 membered cycloalkyl orheterocycloalkyl ring.
 4. The compound or salt of claim 2, wherein atleast one pair of two R² together with the carbon atom to which they areattached form a saturated or unsaturated 5 membered cycloalkyl orheterocycloalkyl ring, wherein the heterocycloalkyl ring has 1 or 2 ringheteroatoms selected from O, S, and N, and wherein said cycloalkyl andheterocycloalkyl ring are optionally substituted with 1 to 3 R³. 5.(canceled)
 6. (canceled)
 7. (canceled)
 8. (canceled)
 9. The compound orsalt of claim 1, wherein at least one R² has a structure:

W is O, S, or NR⁴; and Q is selected from the group consisting of C₁₋₆alkyl, C₂₋₆ alkenyl, C₂₋₆ alkynyl, N(R⁴)₂, and OR⁴.
 10. The compound orsalt of claim 9, wherein W is O and Q is OR⁴.
 11. The compound or saltof claim 10, wherein R⁴ is H or C₁₋₆ alkyl.
 12. (canceled)
 13. Thecompound or salt of claim 1, wherein L is selected from the groupconsisting of uninterrupted C₁₋₁₈alkylene,

Y¹, Y², and Y³ are each independently O or NR⁴, and

indicates that the double bond is cis or trans.
 14. (canceled)
 15. Thecompound or salt of claim 1, wherein L is selected from the groupconsisting of C₁alkylene, C₂alkylene, C₃alkylene, C₄alkylene,C₅alkylene, C₆alkylene, C₇alkylene, C₈alkylene, C₉alkylene, C₁₀alkylene,C₁₁alkylene, and C₁₂alkylene.
 16. (canceled)
 17. (canceled)
 18. Thecompound or salt of claim 13, wherein at least one of Y¹ and Y² is O.19. (canceled)
 20. The compound or salt of claim 13, wherein at leastone of Y¹ and Y² is NR⁴.
 21. (canceled)
 22. The compound or salt ofclaim 13, wherein R⁴ is H or C₁₋₆ alkyl.
 23. (canceled)
 24. The compoundor salt of claim 1, wherein L is C₂₋₁₈alkenylene.
 25. (canceled) 26.(canceled)
 27. (canceled)
 28. (canceled)
 29. The compound or salt ofclaim 1, wherein L is C₁₋₁₈alkylene or C₂₋₁₈alkenylene interrupted by atleast one phenyl.
 30. (canceled)
 31. The compound or salt of claim 13,wherein Y³ is O.
 32. The compound or salt of claim 13, wherein Y³ isNR⁴.
 33. (canceled)
 34. (canceled)
 35. The compound or salt of claim 1,wherein L is


36. The compound or salt of claim 35, wherein R⁴ is C₂₋₆ alkynyl. 37.(canceled)
 38. (canceled)
 39. (canceled)
 40. A method of treating orpreventing a disease or disorder capable of being modulated by NFκBpathway inhibition, comprising administering to a subject in needthereof a therapeutically effective amount of the compound or salt ofclaim
 1. 41. The method of claim 40, wherein the disease or disorder isselected from the group consisting of cancer, autoimmune diseases,inflammatory diseases, diabetes, cardiovascular diseases, andneurological diseases.
 42. (canceled)